This invention relates to perfusable cell culture device, and more particularly comprises a flow divider membrane attached to a lower polymeric layer in a manner which provides channels for uniform fluid distribution over the entire surface of the flow divider membrane and into a bed of cells dispersed thereon.
Early in the history of tissue culture a need to perfuse culture systems was recognized. As early as 1912, attempts to furnish cultures of animal cells with a continuous supply of fresh nutrient medium were made. Feder, J. , Tolbert, W. R. Mass Culture of Mammalian Cells in Perfusion Systems, ABL, Jan./Feb. 1985, p. 24-36. Since these early attempts, development of perfusion systems has continued and advanced significantly. For a good analysis of the development of perfused systems used for examination of mammalian cells see: Nahapetian, A. T. (1986). "Growth and Maintenance of Anchorage Dependent Mammalian Cells in Perfused Systems and Metabolism of Nutrients", Chapter 7, Mammalian Cell Technology, W. G. Thilly(ed), pp. 151-165, Buttersworth, Boston.
Conventional cell culture systems incorporate a membrane or membranes used to diffuse either nutrients, metabolic waste, gases or cellular products into or out of the cell's environment. For example, U.S. Pat. No. 4,308,351 Leighton et. al. describes an apparatus comprising encasing a tissue sample attached to a membrane and a support for the membrane, in a specially designed closed container filled with solution and then bathed in a nutrient solution. Due to a designed nutrient concentration gradient occurring in the tissue, the tissue sample receives oxygen and nutrients by diffusion through the membrane from the nutrient bath as the membrane is exposed to the bath through apertures in the closed container. U.S. Pat. No. 3,911,140 Osborne et. al. describes a fermenter in which a dialysis membrane separates a culture medium containing propagating microorganisms from a regenerating solution containing nutrients. The dialysis membrane is impermeable to the microorganisms and allows diffusion of desired nutrients based on a concentration gradient of nutrients and lactic acid on either side of the membrane. Contrary to the conventional art, the present invention incorporates a flow divider membrane which due to the manner in which it is affixed to the lower polymeric layer, allows for uniform distribution of culture medium over the entire flat surface of the flow divider membrane, rather than incorporating a filter to control diffusion of nutrients, metabolic waste, gases or cellular product between varying sides of a concentration gradient.
The present invention provides a device designed to perfuse cells during culture which is capable of providing a favorable environment for expansion and maintenance of both anchorage dependent (in combination with microcarriers) and anchorage independent cells, such device having the following characteristics: 1) it provides continuous supply of nutrients and continuous removal of metabolic end products; 2) it provides adequate O.sub.2 and CO.sub.2 exchange with the surrounding environment; 3) it maintains a favorable pH gas exchange; 4) it is designed such that the nutrients flow uniformly through the cell bed, independent of diffusion gradients or thermal agitation; 5) it is transparent thereby allowing visual examination of the culture and additionally allowing light to pass through the vessel thereby providing an essential environment when the device is used for plant cells; 6) it equilibrates temperature easily with the surrounding environment; 7) it exerts no shear, since there is no need for constant mixing of the culture; 8) it provides a sterile environment; 9) it is non-breakable, disposable or sterilizable by autoclave; 10) it is not bulky and can be stacked one on top of another when not in use, therefore conserving space; and 11) it is more cost effective than other perfused vessels available on the market.